Regulation of T-cell receptor Dβ1 promoter by KLF5 through reiterated GC-rich motifs

Abstract

AbstractRearrangement of T-cell receptor (TCR) and immunoglobulin genes by a common V(D)J recombination machinery is regulated by developmentally specific chromatin changes at the target locus, a process associated with transcription. At the TCRβ locus, the Eβ enhancer and the Dβ1 promoter regulate germline transcription originating near the TCR Dβ1 gene segment. The Dβ1 promoter contains 3 GC-rich motifs that bind a common set of nuclear proteins from pro–T-cell lines. Mutations that diminish the binding of nuclear proteins also diminish the activity of the Dβ1 promoter in transcriptional reporter assays. Using a yeast one-hybrid approach, 3 Krüppel-like factors—KLF3, KLF5, and KLF6—and a novel zinc finger protein were identified in a thymus library, all of which bound the GC-rich motif in a sequence-specific manner. Of these genes, KLF5 mRNA was expressed in a restricted manner in lymphoid cells and tissues, with highest expression in pro–T-cell lines and Rag-deficient thymocytes. Antibody supershift studies and chromatin immunoprecipitation assay confirmed that KLF5 bound the Dβ1 promoter. In reporter gene assays, KLF5 but not KLF6 efficiently transactivated the Dβ1 promoter, whereas a dominant-negative KLF5 construct inhibited reporter expression. These data suggest that reiterated GC motifs contribute to germline TCRβ transcription through binding of KLF5 and other Krüppel family members and that restricted expression of KLF5 may contribute to lineage-specific regulation of germline TCRβ transcription.